Regulates Breast Cancer Cell Proliferation Research Articles

ClpP regulates breast cancer cell proliferation, invasion and apoptosis by modulating the Src/PI3K/Akt signaling pathway.

BackgroundCaseinolytic protease P (ClpP), which is located on the inner mitochondrial membrane, degrades mitochondrial proteins damaged by oxidative stress. The role of ClpP varies among tumor types. However, the expression pattern and biological functions of ClpP in breast cancer (BC) have not yet been investigated.MethodsThe Cancer Genome Atlas (TCGA) and Kaplan Meier-plotter database were used to analyze the expression level of ClpP in BC tissues, relationships with clinicopathological characteristics, and the influence on the prognosis of BC. Protein and mRNA expression levels of ClpP in BC cell lines and tissues were detected by quantitative real-time PCR, western blot and immunohistochemical (IHC) analyses. The colony formation assay, transwell assay and flow cytometric analysis were performed to assess various functions of ClpP. Western blot analysis was also conducted to determine the mechanism of ClpP.ResultsClpP expression was markedly increased in BC cells and tissues. High expression of ClpP was significantly correlated with the T stage, estrogen receptor (ER) expression, and poor recurrence-free survival (RFS) in TCGA and Kaplan Meier-plotter database. ClpP silencing significantly inhibited proliferation, migration, invasion, and promoted apoptosis of BC cells, which resulted in suppression of the Src/PI3K/Akt signaling pathway. The gain-of-function assay confirmed partial these results.

MiR‑200b regulates breast cancer cell proliferation and invasion by targeting radixin

Radixin is an important member of the Ezrin-Radixin-Moesin protein family that is involved in cell invasion, metastasis and movement. microRNA (miR)-200b is a well-studied microRNA associated with the development of multiple tumors. Previous bioinformatics analysis has demonstrated that miR-200b has a complementary binding site in the 3'-untranslated region of radixin mRNA. The present study aimed to investigate the role of miR-200b in regulating radixin expression, cell proliferation and invasion in breast cancer. Breast cancer tissues at different Tumor-Node-Metastasis (TNM) stages were collected; breast tissues from patients with hyperplasia were used as a control. miR-200b and radixin mRNA expression levels were tested by reverse transcription-quantitative PCR. Radixin protein expression was detected by western blotting. The highly metastatic MDA-MB-231 cells were divided into four groups and transfected with a miR-negative control (NC), miR-200b mimic, small interfering (si)RNA-NC or siRNA targeting radixin. Cell invasion was evaluated by Transwell assay and cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine staining. Compared with the control group, radixin mRNA expression was significantly higher in breast cancer tissues and increased with TNM stage. miR-200b expression levels exhibited the opposite trend. Radixin mRNA expression in breast cancer cells was notably higher, whereas miR-200b expression was lower compared with that in normal breast epithelial MCF-10A cells. The expression of radixin was higher, whereas miR-200b was lower in MDA-MB-231 cells compared with that in MCF-7 cells. miR-200b mimic or siRNA-radixin transfection downregulated the expression of radixin in MDA-MB-231 cells and attenuated the invasive and proliferative abilities of these cells. miR-200b-knockdown and radixin overexpression were associated with enhanced cell invasion in breast cancer. In conclusion, miR-200b regulates breast cancer cell proliferation and invasion by targeting radixin expression.

A Long Non-coding RNA Lnc712 Regulates Breast Cancer Cell Proliferation.

Great quantity of intergenic noncoding RNAs (lncRNAs) have been identified in the mammalian genome and involved in various biological processes, especially in the development and metastasis of cancer. In this study, we identified one lncRNA, lncRNA NONHSAT028712 (Lnc712), was highly expressed in breast cancer cell lines and tissues based on microarray screening. Knockdown of Lnc712 largely inhibited breast cancer cell proliferation. Mechanistically, Lnc712 bound specifically to heat-shock protein 90 (HSP90). Interaction between Lnc712 and HSP90 is required for HSP90 binding to cell division cycle 37 (Cdc37). The Lnc712/HSP90/Cdc37 complex regulated cyclin-dependent kinase 2 (CDK2) activation and then triggered breast cancer cell proliferation. In summary, our results identified a new lncRNA regulate breast cancer proliferation though interaction with HSP90.

UBE2O promotes the proliferation, EMT and stemness properties of breast cancer cells through the UBE2O/AMPK\u03b12/mTORC1-MYC positive feedback loop

Ubiquitin-conjugating enzyme E2O (UBE2O) is a large E2 ubiquitin-conjugating enzyme that possesses both E2 and E3 ligase activities. Ectopic UBE2O overexpression is associated with a variety of human diseases, especially cancers. However, the expression profile and functional biology of UBE2O in human breast cancer (BC) remain unclear. In this study, we found that UBE2O was significantly overexpressed in human BC tissues and cells. Patients with high UBE2O expression tended to have a high risk of metastasis and poor prognosis. In vitro assays revealed that UBE2O promoted BC cell proliferation and epithelial–mesenchymal transformation (EMT) and endowed BC cells with cancer stemness properties (CSPs). UBE2O knockdown in MDA-MB-231 cells suppressed tumour growth and lung metastasis in MDA-MB-231 xenograft mouse models. Mechanistically, UBE2O functioned as a ubiquitin enzyme of AMPKα2, promoting its ubiquitination and degradation and thus activating the mTORC1 signal pathway and contributing to BC oncogenesis and metastasis. Furthermore, as a downstream factor of the UBE2O/AMPKα2/mTORC1 axis, the oncoprotein MYC transcriptionally promoted UBE2O and formed a positive feedback loop in human BC. Collectively, our study demonstrated that UBE2O/AMPKα2/mTORC1-MYC forms a positive feedback loop in human BC cells that regulates BC cell proliferation and EMT and endows BC cells with CSPs.

MiR-519 regulates the proliferation of breast cancer cells via targeting human antigen R.

Breast cancer is one of the most prevalent types of cancer among women that leads to millions of deaths worldwide every year. The mechanisms of breast cancer pathogenesis remain unclear. It has been reported that aberrant expression of miR-519, is associated with breast cancer development; however, the effects of miR-519 on breast cancer cell proliferation remain unknown. Therefore, the present study aimed to determine whether miR-519 could regulate breast cancer cell proliferation. A total of 20 pairs of primary breast cancer and adjacent normal tissues were collected from patients with breast cancer. miR-519 expression level was determined by reverse transcription-quantitative polymerase chain reaction. Furthermore, miR-519 mimics or inhibitors were transfected into breast cancer MCF-7 cells in order to up- or downregulate miR-519 expression. Subsequently, human antigen R (HUR), BCL-2 and BAX protein levels were analyzed by western blotting. MCF-7 cell proliferation was assessed using MTT and colony formation assays. A luciferase assay was performed to verify whether miR-519 could directly bind to HUR mRNA. The results demonstrated that miR-519 expression level was lower in primary breast cancer tissues compared with adjacent normal tissues. Furthermore, miR-519 overexpression and downregulation inhibited and stimulated MCF-7 cell proliferation, respectively. In addition, the results from luciferase assay demonstrated that HUR was a target of miR-519. HUR overexpression could reverse the effect of miR-519 mimics on MCF-7 cell proliferation, whereas HUR silencing could rescue the effect of miR-519 inhibitors on MCF-7 cell proliferation. These findings suggested that miR-519 may regulate MCF-7 cell proliferation by targeting HUR.

Myoglobin Regulates Breast Cancer Cell Proliferation and Migration Through Modulation of Mitochondrial Fusion and Respiration

Myoglobin Regulates Breast Cancer Cell Proliferation and Migration Through Modulation of Mitochondrial Fusion and Respiration

Abstract 2036: Osteoblasts use multiple mechanisms of cellular communication to regulate breast cancer cell proliferation in bone

Abstract Breast cancer (BC) preferentially metastasizes to bone. While the mechanism for preferred metastasis is unknown, bone likely provides a hospitable environment that attracts BC cells (BCCs) and allows them to colonize and grow. Bone metastases perturb the balance of normal bone remodeling. Emerging evidence suggests that OBs may be altered and respond differently to cancer cells during metastatic BC progression. According to one study, OBs suppress cancer cell growth in early metastasis. Our work suggests OBs reduce BCC growth in bone prior to macrometastatic colonization. Thus, our data suggest OBs have tumor-inhibitory properties. We have new evidence to suggest that communication between OBs and BCCs ‘educates’ OBs to produce factors that suppress BCC proliferation in bone. We have in-vitro and in-vivo mouse-model evidence that ‘educated’ osteoblasts (EOs) have a unique secretory protein profile compared to ‘uneducated’ OBs. We also identified EOs as being present in the bone tissue samples of human patients with bone metastatic BC via multi-plex immunofluorescence. When we treated BCCs with EO conditioned media (CM), BCC proliferation was reduced in both triple negative and ER+ metastatic BCCs, while CM from ‘uneducated’ OBs did not affect BCC proliferation. This effect was mediated through alterations in EO production of decorin and NOV. We identified EO CM as a rich source of exosomes (exo) and confirmed the presence of an exo population via iodixanol density gradient and western blotting for specific exo protein markers. We found that EO-derived exo, but not ‘uneducated’ OB-derived exo, decreased proliferation of ER+ and triple negative BCC. Also, exposure to EO-derived exo 1) decreased the number of Ki67 positive metastatic BCC, and 2) decreased the number of metastatic BCC in the S phase of cell cycle as measured by EdU incorporation. Moreover, we labeled EO exo with RFP-conjugated CD63 to visually confirm exo transfer from EO cells to BCCs using confocal microscopy. And, co-culture with EOs increased triple negative and ER+ metastatic BC expression of p21 compared to co-cultures with ‘uneducated’ OBs. Our data suggest that EOs use multiple mechanisms of cellular communication to regulate BCC proliferation in bone. Impact: Our data suggest OBs produce factors that suppress metastatic BCC growth. Much less attention has been given to OB interactions with tumor cells at sites of bone metastasis due to observations that OB populations are reduced at sites of advanced osteolysis. However, we propose that OBs may be valuable endogenous targets to aid in suppression of metastatic BC growth in the niche in concert with therapeutic drugs to kill the cancer cells. Our data suggest there is a population of OBs that demonstrate a functional role in suppressing metastatic BCC growth; a property capable of exploitation. For these reasons, OBs and EOs are strong candidates for therapeutic targeting. Citation Format: Alexus D. Kolb, Alison B. Shupp, Dimpi Mukhopdhyay, Karen M. Bussard. Osteoblasts use multiple mechanisms of cellular communication to regulate breast cancer cell proliferation in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2036.

Abstract P5-05-12: Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1

Abstract Introduction: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the Progesterone Receptor Membrane Component 1 (PGRMC1), a heme – binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. Some evidence suggests that progesterone can stimulate and regulate breast cancer cell proliferation. The basis of the signaling mechanisms by which Progesterone exerts its function remains largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancer and both could become viable therapeutic options. We, aim to investigate the role of PGRMC1 in progesterone driven breast cancers. Materials and Methods: Human breast tissues were utilized to identify PGRMC1 expression along with a panel of normal and breast cancer cell lines. Two breast cancer cell lines (ZR-75-1 and MDA-MB-468) were selected and treated with progesterone and AG-205 (PGRMC1 inhibitor) at different concentrations to assess optimum dosage. We performed MTS assay, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry for measuring cell proliferation, apoptosis and key markers involved in these processes. We also performed an in silico analysis to compare the expression of PGRMC1 in various cell lines and breast cancer tissues. Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Progesterone treatment increased cell proliferation in a dose dependent manner while AG-205 decreased cell proliferation in a dose dependent manner in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in normal breast epithelial cells. AG-205 also, induced apoptosis in both ZR-75-1 and MDA-MB-468 cell lines. Furthermore, short-term treatment of progesterone increased both mRNA and protein expression of PGRMC1. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax, Bim) revealed that PGRMC1 facilitated the proliferative effect of progesterone. Interestingly progesterone increases phosphorylation of EGFR and treatment of AG-205 alters EGFR expression in a dose dependent manner. Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in regulating progesterone driven cell proliferation in both ER-positive and triple negative breast cancer cells. These initial findings uncover the potential of PGRMC1 as a therapeutic target for breast cancers. Citation Format: Pedroza DA, Subramani R, Galvez A, Lakshmanaswamy R. Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-12.

Protein carbonylation as a possible way to modulate breast cancer cell proliferation

Introduction.High rates of cancer incidence and mortality worldwide dictate the necessity of developing new methodological approaches in understanding the molecular mechanisms of cancer progression associated with intracellular redox regulation imbalance.The objectiveof the study was to evaluate the role of protein carbonylation in regulating breast cancer cell proliferation under redox status modulation.Materials and Methods. In the intact breast cancer cells and in the cells cultured under redox status modulation using 5mM N-ethylmaleimide (an - SH group blocker) and 5 Mm 1,4-dithioerythritol (a thiol group protector), the concentration of thioredoxin and its carbonylated form was measured using Western blot analysis. The activity of thioredoxin reductase and the level of protein carbonyl derivatives were determined using spectrophotometry. Cell cycle phase distribution was evaluated by flow cytometry.Results and Discussion. Under the effect of N-ethylmaleimide, cell cycle arrest in the S-phase was confirmed by oxidative modification of proteins, including thioredoxin carbonylation. When culturing MCF-7 cells in the presence of 1,4-dithioerythritol, cell cycle arrest in the G0/G1 phases was associated with a rise in the concentrations of reduced thioredoxin and glutathione forms.Conclusion.The thioredoxin system and oxidative modification of proteins are involved in redox-dependent modulation of breast cancer cell proliferation. Studies in the area of redox proteomics offer great potential to seek molecular targets of malignant transformation of breast cells.

MiR-34b/c-5p and the neurokinin-1 receptor regulate breast cancer cell proliferation and apoptosis.

ObjectivesMiR‐34 is a tumour suppressor in breast cancer. Neurokinin‐1 receptor (NK1R), which is the predicted target of the miR‐34 family, is overexpressed in many cancers. This study investigated the correlation and clinical significance of miR‐34 and NK1R in breast cancer.Materials and MethodsWestern blotting, quantitative reverse transcription‐PCR (qRT‐PCR) and luciferase assays were conducted to analyse the regulation of NK1R by miR‐34 in MDA‐MB‐231, MCF‐7, T47D, SK‐BR‐3 and HEK‐293 T cells. MiR‐34b/c‐5p, full‐length NK1R (NK1R‐FL) and truncated NK1R (NK1R‐Tr) expression in fifty patients were quantified by qRT‐PCR and correlated with their clinicopathological parameters. CCK‐8 assays, colony formation assays and flow cytometry were used to measure cell proliferation and apoptosis in MDA‐MB‐231 and MCF‐7 cells transfected with miR‐34b/c‐5p or NK1R‐siRNA and before treatment with or without Substance P (SP), an endogenous peptide agonists of NK1R. The effect of NK1R antagonist aprepitant was also investigated. In vivo xenograft models were used to further verify the regulation of NK1R by miR‐34b/c‐5p.ResultsExpression levels of miR‐34b/c‐5p and NK1R‐Tr, but not NK1R‐FL, were associated with enhanced malignant potential, such as tumour stage and Ki67 expression. The overexpression of miR‐34b/c‐5p or NK1R silencing potently suppressed cell proliferation and induced G2/M phase arrest and the apoptosis of MDA‐MB‐231 and MCF‐7 cells. The NK1R antagonist aprepitant had similar effects. In vivo studies confirmed that miR‐34b/c‐5p overexpression or NK1R silencing reduced the tumorigenicity of breast cancer. In addition, SP rescued the effects of miR‐34b/c‐5p overexpression or NK1R silencing on cell proliferation and apoptosis in vitro and in vivo assays.ConclusionsMiR‐34b/c‐5p and NK1R contribute to breast cancer cell proliferation and apoptosis and are potential targets for breast cancer therapeutics.

MicroRNA-421-targeted PDCD4 regulates breast cancer cell proliferation.

MicroRNAs (miRNAs) are expressed aberrantly in various types of cancer, and negatively regulate the expression of target genes which may be useful in therapeutic strategies in several biological processes. In the present study, the expression levels and the effects of miRNA (miR)-421 in breast cancer tissues and MCF-7 and MDA-MB-231 cells were evaluated to elucidate therapeutic targets in breast cancer cells. The putative targets of miR-421 were predicted by bioinformatics approaches, and the expression levels of miR-421 were measured in MCF-7 and MDA-MB-231 cells by reverse transcription-quantitative polymerase chain reaction analysis following miR-421 knockdown. The rates of cell proliferation, migration capacity, invasiveness and apoptosis were determined in miR-421 inhibitor-transfected MCF-7 and MDA-MB-231 cells. The expression levels of target proteins regulated by miR-421 in MCF-7 and MDA-MB-231 cells were analyzed by western blot analysis. miR-421 was increased significantly in breast cancer tissues and cells, and was regulated by miR-421 antisense oligonucleotides. The knockdown of miR-421 in MCF-7 and MDA-MB-231 cells decreased cell proliferation, migration capacity and invasiveness, and promoted apoptosis compared with control groups. The expression of target protein programmed cell death 4 (PDCD4) were decreased in MCF-7 and MDA-MB-231 cells transfected with miR-421 inhibitors. These results suggested a correlation between miR-421 and PDCD4, and physiological functions of breast cancer cells, suggesting that miR-421 may be a potential strategy in the therapy of breast cancer.

MYBL2 Is Targeted by miR-143-3p and Regulates Breast Cancer Cell Proliferation and Apoptosis.

Breast cancer remains a public health issue on a global scale. The present study aimed to explore the functional role of MYB proto-oncogene like 2 (MYBL2) in breast cancer, as well as underlying mechanisms. The regulatory relationship between miR-143-3p and MYBL2 was analyzed, and the effects of dysregulation of miR-143-3p and MYBL2 on cell proliferation and apoptosis were investigated. The results showed that MYBL2 and miR-143-3p were inversely expressed in breast cancer tissues and cells: MYBL2 was highly expressed, whereas miR-143-3p was lowly expressed. MYBL2 was confirmed as a target gene of miR-143-3p. Suppression of MYBL2 inhibited proliferation and induced apoptosis of breast cancer cells, which was similar to the effects of overexpression of miR-143-3p. Our findings reveal that MYBL2 is targeted by miR-143-3p and regulates breast cancer cell proliferation and apoptosis.

Abstract 108: Abi1 positively regulates lung metastasis of aggressive breast cancer in PyMT mouse model

Abstract Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women and is estimated to cause 40,000 deaths this year. Despite standard of care, breast cancer patients often relapse after a few years of treatment thus highlighting the need for new molecular targets for improved management of metastatic disease. Abelson interactor 1 (Abi1) is an adaptor protein associated with the WAVE (Wiscott-Aldrich syndrome protein family verprolin homologous) regulatory complex and Arp2/3 (Actin-related proteins 2 and 3)-mediated actin cytoskeleton remodeling. Our analysis of human tumor data indicates that Abi1 is frequently upregulated in invasive breast cancers, is associated with poor survival, and may promote an aggressive breast tumor phenotype. Abi1 has been shown to positively regulate breast cancer cell proliferation, motility, division, and invasion in vitro however its exact role in vivo remains unknown. We therefore hypothesize that Abi1 promotes breast tumor progression and metastasis in a mouse breast cancer model. Using the well-characterized Polyoma Middle T (PyMT) breast cancer model, we conditionally deleted Abi1 from the mammary epithelium and determined the effects on tumor growth kinetics and WAVE complex protein levels. Interestingly, our studies show delayed tumor growth only in mice with heterozygous deletion of Abi1 while homozygote KO mice show relatively unchanged tumor growth. Western blot analyses of Abi1 KO breast tumors show concomitant loss of WAVE complex proteins supporting our previous findings that WAVE complex integrity is dependent on Abi1. Our data also show significant upregulation of Abi2 only in homozygous Abi1 KO mice, suggesting a potential compensatory role for Abi2 that may support primary tumor growth in absence of Abi1. Most interestingly, Abi1 null PyMT mice show significantly reduced incidence of lung metastasis, supporting our hypothesis that Abi1 promotes metastasis of breast cancer cells. In summary, Abi1 loss abrogates lung metastasis in PyMT mice however primary tumor growth remains largely unaffected, possibly due to a compensatory mechanism by a related protein, Abi2. This work will establish the biochemical dynamics between members of the Abi protein family and WAVE complex to improve targeted treatments in aggressive human breast cancer. [Supported by NCI grant R01-CA161018 and Carol M. Baldwin Breast Cancer Research Fund of CNY] Citation Format: Angelina Regua, Isabelle Bichindaritz, Tiffany Caza, Julie R. White, Alexander Nappi, Claudia Mondragon, Mira Krendel, Gennady Bratslavsky, Abirami Sivapiragasam, Leszek Kotula. Abi1 positively regulates lung metastasis of aggressive breast cancer in PyMT mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 108.

PSMD2 regulates breast cancer cell proliferation and cell cycle progression by modulating p21 and p27 proteasomal degradation

Alterations in the ubiquitin-proteasome system (UPS) and UPS-associated proteins have been implicated in the development of many human malignancies. In this study, we investigated the expression profiles of 797 UPS-related genes using HiSeq data from The Cancer Genome Atlas and identified that PSMD2 was markedly upregulated in breast cancer. High PSMD2 expression was significantly correlated with poor prognosis. Gene set enrichment analysis revealed that transcriptome signatures involving proliferation, cell cycle, and apoptosis were critically enriched in specimens with elevated PSMD2. Consistently, PSMD2 knockdown inhibited cell proliferation and arrested cell cycle at G0/G1 phase in vitro, as well as suppressed tumor growth in vivo. Rescue assays demonstrated that the cell cycle arrest caused by silencing PSMD2 partially resulted from increased p21 and/or p27. Mechanically, PSMD2 physically interacted with p21 and p27 and mediated their ubiquitin-proteasome degradation with the cooperation of USP14. Notably, intratumor injection of therapeutic PSMD2 small interfering RNA effectively delayed xenograft tumor growth accompanied by p21 and p27 upregulation. These data provide novel insight into the role of PSMD2 in breast cancer and suggest that PSMD2 may be a potential therapeutic target.

Sevoflurane suppresses proliferation by upregulating microRNA-203 in breast cancer cells.

Rapid proliferation is one of the critical characteristics of breast cancer. However, the underlying regulatory mechanism of breast cancer cell proliferation is largely unclear. The present study indicated that sevoflurane, one of inhalational anesthetics, could significantly suppress breast cancer cell proliferation by arresting cell cycle at G1phase. Notably, the rescue experiment indicated that miR-203 was upregulated by sevoflurane and mediated the function of sevoflurane on suppressing the breast cancer cell proliferation. The present study indicated the function of the sevoflurane/miR-203 signaling pathway on regulating breast cancer cell proliferation. These results provide mechanistic insight into how the sevoflurane/miR-203 signaling pathway supresses proliferation of breast cancer cells, suggesting the sevoflurane/miR-203 pathway may be a potential target in the treatment of breast cancer.

MiR-519d-3p suppresses breast cancer cell growth and motility via targeting LIM domain kinase 1.

Breast cancer is the most common female cancer in women, and its estrogen receptor (ER)-negative subtype (ENBC) and triple-negative subtype (TNBC) have unfavorable prognosis in comparison with ER-positive subtype. MiRNAs are small noncoding RNAs that bind to the 3'-UTR region of targeting mRNAs to regulate gene expression. Mir-519d-3p was found to be associated with breast cancer for its potential role in proliferation and metastasis. To explore its potential role and mechanism of miR-519d-3p in breast carcinogenesis, we determined whether miR-519d-3p regulates breast cancer cell proliferation and motility by performing wound-healing assays and migration-invasion assays. We found that miR-519d-3p significantly inhibits proliferation and motility of ENBC and TNBC cells. Overexpression of miR-519d-3p arrested breast cancer cells in the G0/G1 phase and reduced the expression of CDK4, 6/Cyclin D1, and CDK2/Cyclin E1. It was reported that miR-519d-3p or miR-519d-3p expression was associated with cancer metastasis and clinical staging. Since LIM domain kinase 1 (LIMK1) was highly expressed in breast cancer and a major regulator of breast cancer growth and metastasis, we further demonstrated that LIMK1 is a potential target of miR-519d-3p by dual-luciferase report assay. Mir-519d-3p decreases LIMK1 expression at mRNA and protein levels, and the protein level and phosphorylation of cofilin 1 (CFL1), one of the key downstream targets of LIMK1. Our findings suggest that miR-519d-3p regulates the LIMK1/CFL1 pathway in breast cancer and this new venue could be targeted for future breast cancer therapy.

MiR-29a regulated ER-positive breast cancer cell growth and invasion and is involved in the insulin signaling pathway.

Increasing amounts of evidence show that insulin can activate different insulin signaling pathways to promote breast cancer growth and invasion. miR-29a plays crucial roles in decreasing glucose-stimulated insulin secretion, as well as in regulating breast cancer cell proliferation and EMT. However, the mechanism responsible for the regulatory effects of miR-29a on breast cancer growth and invasion and the relationship between these effects and insulin signaling remains unclear. Herein, we showed that human insulin increased miR-29a expression in ER-positive breast cancer cells and that miR-29a facilitated the ability of insulin to promote breast cancer cell proliferation and migration. We found that miR-29a-induced cell proliferation and metastasis acceleration occurred primarily through ERK phosphorylation. The IGF-1R is the upstream target gene of miR-29a, while CDC42 and p85α are the downstream target genes of miR-29a. These results have provided us with information regarding the molecular mechanisms by which hyperinsulinemia promotes breast cancer occurrence and development and thus leads to a poor prognosis in breast cancer patients and indicate that miR-29a plays an important role in breast cancer development and invasion.

Identification of TG100-115 as a new and potent TRPM7 kinase inhibitor, which suppresses breast cancer cell migration and invasion

BackgroundTransient receptor potential melastatin 7 (TRPM7) regulates breast cancer cell proliferation, migration, invasion and metastasis in its ion channel- and kinase domain-dependent manner. The pharmacological effects of TRPM7 ion channel inhibitors on breast cancer cells have been studied, but little is known about the effects of TRPM7 kinase domain inhibitors due to lack of potent TRPM7 kinase inhibitors. MethodsScreening was performed by using TRPM7 kinase assay. Effects of TG100-115 on breast cancer cell proliferation, migration, invasion, myosin IIA phosphorylation, and TRPM7 ion channel activity were assessed by using MTT, wound healing, transwell assay, Western blotting, and patch clamping, respectively. ResultsWe found that CREB peptide is a potent substrate for the TR-FRET based TRPM7 kinase assay. Using this method, we discovered a new and potent TRPM7 kinase inhibitor, TG100-115. TG100-115 inhibited TRPM7 kinase activity in an ATP competitive fashion with over 70-fold stronger activity than that of rottlerin, known as a TRPM7 kinase inhibitor. TG100-115 has little effect on proliferation of MDA-MB-231 cells, but significantly decreases cell migration and invasion. Moreover, TG100-115 inhibits TRPM7 kinase regulated phosphorylation of the myosin IIA heavy chain and phosphorylation of focal adhesion kinase. TG100-115 also suppressed TRPM7 ion channel activity. ConclusionsTG100-115 can be used as a potent TRPM7 kinase inhibitor and a potent inhibitor of breast cancer cell migration. General significanceTG100-115 could be a useful tool for studying the pharmacological effects of TRPM7 kinase activity aimed at providing insight into new therapeutic approaches to the treatment of breast cancer.

PGC-1 alpha interacts with microRNA-217 to functionally regulate breast cancer cell proliferation

BackgroundIn this study, we explored the functional mechanism of PPARg co-activator 1-alpha (PGC-1α) in regulating miR-217-mediated breast cancer development in vitro. MethodsDual-luciferase activity assay was applied to examine the binding of miR-217 on PGC-1α gene. Breast cancer cell lines, MCF-7 and MDA-MB-231 were infected by lentivirus to constitutively downregulate miR-217. Its regulation on PGC-1α expression was investigated by qRT-PCR and western blot. PGC-1α gene was subsequently downregulated by siRNA in miR-217-downregulated breast cancer cells to examine its effect on cancer proliferation and cell-cycle progression. In addition, another downstream target gene of miR-217, DACH1, was further downregulated in breast cancer cells to investigate the functional association of PGC-1α and DACH1 in miR-217-mediated breast cancer regulation. ResultsPGC-1α gene was directly bound by human miR-217. Downregulation of miR-217 in MCF-7 and MDA-MB-231 cells increased PGC-1α production at both mRNA and protein levels. SiRNA-mediated PGC-1α downregulation reversed the inhibition of miR-217-downregulaiton on breast cancer proliferation and cell-cycle progression. Moreover, siRNA-mediated DACH1 downregulation further reversed miR-217-downregulaiton induced inhibition on cancer proliferation and cell-cycle progression in PGC-1α downregulated MCF-7 and MDA-MB-231 cells. ConclusionMiR-217 is the upstream regulator of PGC-1α in breast cancer regulation in vitro, possibly independent of DACH1 signaling pathway.

ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and c-Myc to reduce breast cancer proliferation

MiRNAs are endogenous ~22 nt RNAs which play critical regulatory roles in a wide range of biological and pathological processes, which can act as oncogenes or tumor suppressor genes depending on their target genes. We have recently shown that ANXA1 inhibits the expression of miRNAs including miR196a. Here, we show that miR196a was highly expressed in ER+ MCF-7 breast cancer cells when compared to normal mammary gland cells, with expression levels negatively correlating to ANXA1. ANXA1 inhibits the biogenesis of oncogenic miR-196a by suppressing primary-miR196a indirectly through the stimulation of c-myc and NFkB expression and activity in breast cancer cells. In a negative feedback loop, miR-196a directly inhibits ANXA1 and enhances breast cancer cell proliferation in vitro. Finally, miR196a promotes breast tumor growth in vivo. This study reports a novel regulatory circuit between ANXA1, NF-kB, c-myc and miR-196a which regulates breast cancer cell proliferation and tumor growth.